RESUMO
Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Transcrição Gênica , Animais , Aorta/metabolismo , Apoptose , Diferenciação Celular , Cromatina/metabolismo , Cruzamentos Genéticos , Proteínas de Ligação a DNA/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1(-/-) embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1(-/-) mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1(-/-) embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1(-/-) ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1(-/-) epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1(-/-) cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Transcrição Gênica , Animais , Apoptose/genética , Padronização Corporal/genética , Ciclo Celular/genética , Cruzamentos Genéticos , DNA Ribossômico/genética , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Precursores de RNA/genéticaRESUMO
Extensive chromatin reprogramming occurs at fertilization and is thought to be under the control of maternal factors, but the underlying mechanisms remain poorly understood. We report that maternal Hira, a chaperone for the histone variant H3.3, is required for mouse development past the zygote stage. Male pronucleus formation is inhibited upon deletion of Hira due to a lack of nucleosome assembly in the sperm genome. Hira mutant oocytes are incapable of developing parthenogenetically, indicative of a role for Hira in the female genome. Both parental genomes show highly reduced levels of DNA replication and transcription in the mutants. It has long been thought that transcription is not required for zygote development. Surprisingly, we found that Hira/H3.3-dependent transcription of ribosomal RNA is required for first cleavage. Our results demonstrate that Hira-mediated H3.3 incorporation is essential for parental genome reprogramming and reveal an unexpected role for rRNA transcription in the mouse zygote.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Replicação do DNA/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , RNA Ribossômico/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Replicação do DNA/fisiologia , Fertilização/genética , Fertilização/fisiologia , Chaperonas de Histonas/genética , Histonas/genética , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Zigoto/crescimento & desenvolvimentoRESUMO
Open chromatin is a hallmark of pluripotent stem cells, but the underlying molecular mechanisms are only beginning to be unraveled. In this review we highlight recent studies that employ embryonic stem cells and induced pluripotent stem cells to investigate the regulation of open chromatin and its role in the maintenance and acquisition of pluripotency in vitro. We suggest that findings from in vitro studies using pluripotent stem cells are predictive of in vivo processes of epigenetic regulation of pluripotency, specifically in the development of the zygote and primordial germ cells. The combination of in vitro and in vivo approaches is expected to provide a comprehensive understanding of the epigenetic regulation of pluripotency and reprograming.
